How many genes does a cell need to live?
One answer to that question, researchers are reporting, is 473.
That "minimal genome" -- smaller than the set of genes found in any autonomously replicating cell in nature -- is enough to allow a synthetic bacterium to thrive and multiply in the lab, according to genomics pioneer J. Craig Venter, PhD, of the J. Craig Venter Institute (JCVI) in La Jolla, Calif., and colleagues.
And while the synthetic genome is relatively tiny, it still contains a healthy dollop of mystery -- Venter and colleagues say they don't know what 149 of those genes actually do in the cell, even though they are needed for life and health.
In other words, Venter told reporters in a telephone briefing, "we know about two thirds of the essential biology (and) we're missing a third."
The novel bacterium is dubbed JCVI-syn3.0, or syn 3.0 for short, and builds on earlier work synthesizing the genes of an organism known as Mycoplasma mycoides, one of a genus of bacteria whose genomes are naturally small.
The ability to synthesize the M. mycoides genome -- essentially rebuilding one found in nature -- and use it to power up a cell suggested it might be possible to winnow out non-essential genes and create a bacterium with just those needed to support life, Venter and colleagues noted in Science.
The researchers initially thought the project would be relatively simple, Venter told reporters -- they and others had come up with lists of genes that looked to be essential and they had a tool, called global transposon mutagenesis, that they could use to figure out which other genes were non-essential.
But in fact it took 5 years and several cycles of design, synthesis, and testing to come up with the 473 genes of syn 3.0 and show that they can be used as the operating system for a cell that has the ability to grow and multiply, Venter said.
Outside experts hailed the research as profound and significant, both for the result itself and the technical innovations needed to achieve it.
The Science paper is a "tour de force," commented Sriram Kosuri, ScD, of the University of California Los Angeles.
"During the course of this work they've had to develop several new technologies in genome synthesis and transplantation, as well as hit upon biology that we are yet to learn about," Kosuri said in a statement.
But he cautioned that new fundamental insights or important industrial and medical applications are not likely to come immediately. "That said, they've created a self-replicating biological organism that might be a better starting point for such scientific and engineering goals than continuing to study natural systems," Kosuri said.
Back to Drawing Board
The initial approach in 2010, Venter said, was simply to build a genome based on the "existing knowledge of biology" at the time. "Every one of our designs failed," he said.
So the researchers went back to the drawing board.
First, they divided the genome of M. mycoides into eight segments in such a way that they could be manipulated and studied separately.
Then they tested each segment systematically -- removing gene after gene and then putting the altered segment back into a genome whose other seven-eighths were unaltered. If they removed gene X, for instance, and the new genome failed to work, gene X was deemed essential, while if it did work, the gene was regarded as non-essential.
But genomes based on that division of labor also didn't work, Venter said. It turned out that some genes were "quasi-essential" -- they weren't strictly necessary but if they were absent the organism grew less well.
And other genes formed "lethal pairs" -- either one could be removed, but not both.
"The whole idea of a minimal genome is not as clear-cut as it seemed initially," commented co-author Clyde Hutchison III, PhD, also of the JCVI.
It turned out that some of the quasi-essential genes had to be included if the researchers wanted a bacterium that grew fast enough to be useful in the lab, he said.
But in the end, the researchers were able to eliminate 428 genes from the synthetic M. mycoides genome of 2010, leaving just 473 in syn 3.0.
Unnatural Genome
The final genome of syn 3.0, however, doesn't look much like a natural genome; Venter and colleagues have tidied it up, reorganizing the genes so that similar functions are grouped together.
"This concept of reorganizing what's already there to make it simpler to understand and easier to manipulate is a new thing," commented Christopher Voigt, PhD, of the Massachusetts Institute of Technology in Cambridge, Mass.
Natural genomes are complex and apparently haphazard and it hasn't been clear whether that complexity is essential for life. "If human designers can create an ordered, structured alternative to how life is found in nature, that would speak to the complexity of biology simply being an artifact of how it was shaped by evolution," Voigt said in a statement.
The researchers noted that the minimal genome in this case would not be a minimal genome for other creatures.
The genomes of mycoplasma species are relatively simple to begin with because they live in the rich and stable environments of animal cells and so over the ages they have lost genes they might once have needed.
The new synthetic genome, syn 3.0, is well adapted for life in a research lab, where all its needs are met, while the minimal genome for a creature that had to perform photosynthesis, for example, would be more complicated, Venter said.
While the researchers don't know the exact function of 149 of the genes in syn 3.0, co-author Hutchison said that's slightly misleading.
"Quite a few of those we have rough idea of what they do," he said, but genes fell into the "unknown" category if any part of their function was unclear.
"We might know a gene is part of a transporter in the cell membrane to move some small molecule into the cell," he said, "but if we don't know what small molecule it moves we're putting it in our not-precisely-defined category."
Still Much to Learn
Nonetheless, the knowledge gap illustrates "how much we don't know, even about the core sections of the genome. That is exciting, scientifically," commented Adam Arkin, PhD, of the University of California Berkeley.
The research "presents a moment for reflection about how we're going to approach a better understanding of life," Arkin said in a statement.
The research is an "impressive feat," commented Takanari Inoue, PhD, of Johns Hopkins School of Medicine in Baltimore.
But the minimal genome is "probably only sufficient for survival in an optimal environment." Inoue said in a statement. "It remains untested whether these chimeras can evolve to fit into a new environment -- a capability that is found in most organisms to some degree."
And, he added, "bioethical issues should not be neglected."
"For example, the current study introduces an artificial genome into an existing bacterium without any apparent safeguard mechanisms in place," Inoue said. "Since there are still many factors in the bacterium that are not under control by scientists, it is unclear if we fully understand how the sum of these two components (natural and synthetic) will behave."
The work was funded by Synthetic Genomics and the Defense Advanced Research Projects Agency. Several co-authors, including Venter, are employees of Synthetic Genomics.
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